Physiological active substances such as various hormones and neurotransmitters regulate the biological function via specific receptor proteins present on cell membranes. Many of these receptor proteins are coupled with guanine nucleotide-binding protein (hereinafter sometimes simply referred to as G protein) and mediate the intracellular signal transduction via activation of G protein. These receptor proteins possess the common structure containing seven transmembrane domains and are thus collectively referred to as G protein-coupled receptors or seven-transmembrane receptors (7TMR).
G protein-coupled receptor proteins present on the cell surface of each functional cell and organ in the body, and play important physiological roles as the target of the molecules that regulate the functions of the cells and organs, e.g., hormones, neurotransmitters, physiologically active substances and the like. Receptors transmit signals to cells via binding with physiologically active substances, and the signals induce various reactions such as activation and inhibition of the cells.
To clarify the relationship between substances that regulate complex biological functions in various cells and organs, and their specific receptor proteins, in particular, G protein-coupled receptor proteins, would elucidate the functional mechanisms in various cells and organs in the body to provide a very important means for development of drugs closely associated with the functions.
For example, in various organs, their physiological functions are controlled in vivo through regulation by many hormones, hormone-like substances, neurotransmitters or physiologically active substances. In particular, physiologically active substances are found in numerous sites of the body and regulate the physiological functions through their corresponding receptor proteins. However, it is supposed that many unknown hormones, neurotransmitters or many other physiologically active substances still exist in the body and, as to their receptor proteins, many of these proteins have not yet been reported. In addition, it is still unknown if there are subtypes of known receptor proteins.
It is very important for development of drugs to clarify the relationship between substances that regulate elaborated functions in vivo and their specific receptor proteins. Furthermore, for efficient screening of agonists and antagonists to receptor proteins in development of drugs, it is required to clarify functional mechanisms of receptor protein genes expressed in vivo and express the genes in an appropriate expression system.
In recent years, random analysis of cDNA sequences has been actively studied as a means for analyzing genes expressed in vivo. The sequences of cDNA fragments thus obtained have been registered on and published to databases as Expressed Sequence Tag (EST). However, since many ESTs contain sequence information only, it is difficult to predict their functions from the information.
As one of orphan G protein-coupled receptor proteins, human FPRL1 is known (J. Biol. Chem. 267(11), 7637–7643 (1992)). Agonists of FPRL1 which have been reported include bacterium-derived fMLF, a partial peptide of HIV-derived gp41 or gp120, a partial peptide of prion, intrinsic substances such as Aβ42, a partial peptide of Annexin I and partial peptides of acute phase protein, hCAP18 and NADH dehydrogenase, and lipoxin A4 as lipid (Immunopharmacol. 2, 1–13, 2002).
Alzheimer's disease is a typical nerve degeneration disease involving progressive dementia and loss in recognition ability, and no effective therapy for this disease has been found. As a matter of course, Alzheimer's disease is the most important disease at present in the advent of an aging society, and development of therapeutic agents therefor has a significantly great meaning medically and economically.
Recently, Hashimoto et al. paid attention to the fact that there are fewer lesions in the occipital lobe of a patient with Alzheimer's disease, and by the “death trap” method (L. D' Adamio et al., Semin. Immunol., 9, 17–23, 1997), a gene inhibiting the apotosis of nerve cells into which a causative gene of familial Alzheimer's disease had been introduced was cloned from the occipital lobe (Proc. Natl. Acad. Sci. USA, 98, 6336–6341, 2001). This gene, named humanin (WO 01/21787), encodes a peptide consisting of 24 residues, and a synthetic humanin peptide inhibited not only death of nerve cells into which the familial Alzheimer's disease gene had been introduced, but also nerve apotosis induced by adding β-amyloid also supposed to cause Alzheimer's disease. Humanin is secreted outside of cells and considered to act on nerve cells to inhibit apotosis, but its receptor has not been revealed.
It is reported that Aβ42 is an agonist of FPRL1 and shows chemotaxis via FPRL1, and also that FPRL1 is accumulated in senile spots as a lesion characteristic of Alzheimer's disease. From these findings, the relationship between FPRL1 and inflammatory reaction observed in Alzheimer's disease is suggested (The Journal of Neuroscience, 2001, Vo. 21 RC123).
It is also reported that Aβ42 is incorporated via FPRL1 into macrophage cells thereby forming fibrin agglutination (amyloid-like deposition) (The FASEB Journal, Vol. 15, 2454–2462, November 2001).
Mouse FPRL2 is also known as one of orphan G protein-coupled receptor proteins (Genomics 13(2), 437–440 (1992)).
It is reported that although human FPRL2 is highly homologous to FPR1 that is a receptor of FMLF (formyl-Met-Leu-Phe), human FPRL2 does not react with fMLF. It is also reported that FPRL2 was recognized to be expressed in monocytes, but not recognized to be expressed in neutrophils in which expression of FPR1 and FPRL1 was recognized (Biochem. Biophys. Res. Commun., 201(1), 174–9, May 30, 1994).
It is reported that W-Peptide (Trp-Lys-Tyr-Met-Val-Met-NH2) is an agonist of FPRL1 and FPRL2, and that FPRL2 is highly expressed in monocytes (J. Biol. Chem. 276(24), 21585–21593 (2001)).
It is reported that Helicobacter pylori-derived peptide Hp (2-20) is an agonist of FPRL2 and activates monocytes via FPRL1/FPRL2 (J. Clin. Invest., 108(8), 1221–8, October 2001).
It is reported that FPRL2 whose functions are maintained is expressed in one kind of antigen presenting cells, that is, dendritic cells (mature type, immature type) and considered to regulate trafficking of the dendritic cells (J. Leukoc. Biol., 72(3), 598–607, September 2002).
It is described that rat humanin has a protective activity on nerves (The FASEB Journal, Vol. 16, 1331–1333, August 2002).
Substances that inhibit binding between G protein-coupled proteins and physiologically active substances (i.e., ligands) and substances that bind and induce signals similar to those induced by physiologically active substances (i.e., ligands) have been used as pharmaceuticals, as antagonists and agonists specific to the receptors, that regulate the biological functions. Therefore, discovery and gene cloning (e.g., cDNA) of a novel G protein-coupled receptor that can be targeted for pharmaceutical development are very important means in search for a specific ligand, agonist, and antagonist of the novel G protein-coupled receptor.
However, not all G protein-coupled receptors have been discovered. There are unknown G protein-coupled receptors and many of these receptors in which the corresponding ligands are yet unidentified are called orphan receptors. Therefore, search and functional elucidation of a novel G protein-coupled receptor is awaited.
G protein-coupled receptors are useful in searching for a novel physiological active substance (i.e., ligand) using the signal transduction activity as the index and in search for agonists and antagonists of the receptor. Even if no physiological ligand is found, agonists and antagonist of the receptor may be prepared by analyzing the physiological action of the receptor through inactivation experiment of the receptor (knockout animal). Ligands, agonists, antagonists, etc. of the receptor are expected to be used as prophylactic/therapeutic and diagnostic agents for diseases associated with dysfunction of the G protein-coupled receptor.
Lowering or accentuation in functions of the G protein coupled receptor due to genetic aberration of the receptor in vivo causes some disorders in many cases. In this case, the G protein coupled receptor may be used not only for administration of antagonists or agonists of the receptor, but also for gene therapy by transfer of the receptor gene into the body (or some specific organs) or by introduction of the antisense nucleic acid of the receptor gene into the body (or the specific organ). In the gene therapy, information on the base sequence of the receptor gene is essentially required for investigating deletion or mutation in the gene. The receptor gene is also applicable as prophylactic/therapeutic and diagnostic agents for diseases associated with dysfunction of the receptor.
The present invention relates to determination of a ligand to an orphan G protein-coupled receptor protein FPRL1 or FPRL2 and use of FPRL1 or FPRL2 and its ligand humanin. That is, the object of the present invention is to provide a method of screening a compound (antagonist, agonist) or its salt that alters the binding property between humanin and FPRL1 or FPRL2, a kit for this screening, a compound (antagonist, agonist) or its salt that alters the binding property between humanin and FPRL1 or FPRL2, which is obtainable using the screening method or the screening kit, a pharmaceutical preparation comprising a compound (antagonist, agonist) or its salt that alters the binding property between humanin and FPRL1 or FPRL2 or a compound or its salt that alters the amount of FPRL1 or FPRL2 expressed, etc.